Protein analyses often begin with electrophoretic separation of a mixture of proteins. In a typical application, proteins migrate through a gel under the influence of an electric field. The rate of migration is dependent on the charge, size and shape of the protein. When the separation is complete, blots, on protein-binding membranes are made from the gels on which the electrophoretic separation is performed.
The general techniques for protein separation and for blotting onto a protein-binding membrane are well known (Techniques in Molecular Microbiology, J. Walker and W. Gaastre (Eds); G. Bers and D. Garfin, Bio Techniques, Vol. 3, No. 4, pp. 276-288 (1985)).
“Protein blotting” is a term that refers to the transfer of electrophoretically-resolved protein samples to a protein-binding membrane prior to analysis for the presence of protein by a detection procedure such as immunodetection. Currently, protein-binding membranes include three main compositions that are used for protein blotting: nitrocellulose, nylon and polyvinylidene difluoride (PVDF). Other protein-binding membranes are also available.
After the blotting process, the protein bound to the membrane is visualized via any of a variety of techniques such as for example selective dyeing, radioactive marking, fluorescence, and chemiluminescence.
In immunodetection, a blot is subjected to a general protein stain such as amido black 10b or ponceau S (Nakamura et al., Anal. Biochem. 148: 311-319, 1985), coomassie brilliant blue R-250 (Burnette, Anal. Biochem. 112: 195-203, 1981), India ink (Hancock and Tsang, Anal. Biochem. 133: 157-162, 1983) or colloidal gold (Moeremans et al., Anal. Biochem. 145: 315-321, 1985; Dunn, Methods in Mol. Biol. 112: 319-329, 1999). A separate blot is often then used for the detection of immunoreactive proteins. Side-by-side comparison of the two blots reveals the general location of the immunogens.
Of the general protein stains, amido black 10b, ponceau S and coomassie blue have the advantage of involving relatively quick procedures, requiring about 10-20 minutes operation time. However these stains are characterized by low sensitivity, having a detection limit of about 50-100 ng of protein. India ink has a sensitivity limit of about 50 ng of protein. Staining with India ink requires about four hours to complete. Colloidal gold stain has the highest sensitivity (about 2 nanograms on PVDF membranes). However incubation times of several hours are required for staining with colloidal gold.
Staining efficiency with the stains described above is dependent on membrane composition. In particular, nylon membranes are incompatible with these stains because reactivity of the membrane itself generates a high staining background (Pluskal et al., Biotechniques, 4: 272-282, 1986). In addition, amido black 10b and coomassie blue, though often used for their ease of removal, are not easily removed from PVDF membranes. High background levels of these dyes remain even after extensive washing steps. An additional membrane limitation is that nitrocellulose membranes cannot be used in some staining procedures because of incompatibility with high concentrations of organic solvents such as methanol.
In order to perform additional characterization of a protein following visualization, the protein should remain essentially intact, i.e., retain tertiary structure, characteristic antigenic activity, enzymatic activity, etc, through staining, destaining and stain removal processes. In particular, harsh conditions are required to apply and remove colloidal gold (Magi et al., Methods in Mol. Biol. 112:431-443, 1999). Such harsh conditions often degrade the analyte proteins and thus preclude additional characterization that requires retention of the intact tertiary protein structure.
Thus, in current practice, staining procedures for proteins often involve reagent profiles that are undesirable for reasons including: low sensitivity; lengthy incubation time; reagents that denature or otherwise degrade the analyte proteins precluding further characterization such as immunodetection; and incompatibility with certain protein-binding membrane compositions.
What is needed is a stain and a staining procedure which:
(a) is as rapid as ponceau S or amido black 10b staining, i.e., requiring less than ten minutes;
(b) employs a dye that is easily removed from the protein-binding membrane in destaining, leaving levels of background stain that do not interfere with protein detection;
(c) has a detection limit similar to that achieved by colloidal gold, i.e., capable of allowing detection of proteins down to the nanogram range;
(d) employs reagents and conditions that allow staining and destaining without denaturation of the analyte proteins, thus preserving the characteristic antigenic activity, enzymatic activity, etc.;
(e) is reversible under conditions which do not denature or otherwise degrade the protein analyte and thus preserve the protein's characteristic antigenic activity, enzymatic activity, etc.; and
(f) is broadly applicable to different protein substrates and to different protein-binding membranes.